PMID:16489762

Citation	Bellini, P and Hemmings, AM (2006) In vitro characterization of a bacterial manganese uptake regulator of the fur superfamily. Biochemistry 45:2686-98
Abstract	Fur proteins generally act as negative transcriptional regulators by binding to target regulatory sequences (fur boxes) in the promoter regions of iron-responsive genes. Recently, Rhizobium leguminosarum was reported to contain a protein (Mur(Rl)) of Fur-like sequence, which, under manganese-replete conditions in its native background, repressed transcription of an ABC-type Mn(II) transporter by binding to two nonpalindromic mur boxes in its promoter region. Mur(Rl) displays apparently unusual regulatory flexibility in that it can also repress iron-responsive genes in Escherichia coli under iron-replete conditions. In this study, we quantify the affinities for binding a number of first-row transition-metal cations by Mur(Rl) and demonstrate that, in a fashion similar to E. coli Fur, Mur(Rl) binds Mn(II), Fe(II), Zn(II), and Co(II) with similar micromolar-order dissociation constants. In contrast to the vast majority of Fur proteins, however, Mur(Rl) lacks any high-affinity structural Zn(II) sites. Furthermore, we show that holoMur(Rl) binds as one and two homodimers to both mur and fur boxes in a concentration-dependent fashion in the presence of not only Mn(II) and Fe(II) but also Zn(II) and Co(II). We have developed an analytical method for determination of the individual dissociation constants and find that the DNA-binding affinities are essentially independent of the metal co-effector. These results complement those obtained in vivo by other authors and suggest that the Fur-like protein of R. leguminosarum, a competent ferric uptake regulator in E. coli, is insufficiently discriminating in its metal-binding characteristics to function as a regulator of iron homeostasis in its native background.
Links	PubMed Online version:10.1021/bi052081n
Keywords	ATP-Binding Cassette Transporters/chemistry; ATP-Binding Cassette Transporters/genetics; ATP-Binding Cassette Transporters/metabolism; Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Cation Transport Proteins/chemistry; Cation Transport Proteins/genetics; Cation Transport Proteins/metabolism; Cations, Divalent/metabolism; DNA/metabolism; Edetic Acid/metabolism; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Homeostasis; Iron/metabolism; Kinetics; Ligands; Manganese/metabolism; Manganese/pharmacology; Molecular Sequence Data; Protein Binding; Repressor Proteins/chemistry; Repressor Proteins/genetics; Repressor Proteins/metabolism; Rhizobium leguminosarum/metabolism; Sequence Alignment; Zinc/metabolism
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